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Complete maturation of the plastid protein translocation channel requires a type I signal peptidase.

Inoue K, Baldwin AJ, Shipman RL, Matsui K, Theg SM, Ohme-Takagi M - J. Cell Biol. (2005)

Bottom Line: Next, we show that disruption of a gene encoding plastidic SPase I (Plsp1) resulted in the accumulation of immature forms of Toc75, severe reduction of plastid internal membrane development, and a seedling lethal phenotype.These phenotypes were rescued by the overexpression of Plsp1 complementary DNA.Plsp1 appeared to be targeted both to the envelope and to the thylakoidal membranes; thus, it may have multiple functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Sciences, College of Agricultural and Environmental Sciences, University of California, Davis, CA 95616, USA. kinoue@ucdavis.edu

ABSTRACT
The protein translocation channel at the plastid outer envelope membrane, Toc75, is essential for the viability of plants from the embryonic stage. It is encoded in the nucleus and is synthesized with a bipartite transit peptide that is cleaved during maturation. Despite its important function, the molecular mechanism and the biological significance of the full maturation of Toc75 remain unclear. In this study, we show that a type I signal peptidase (SPase I) is responsible for this process. First, we demonstrate that a bacterial SPase I converted Toc75 precursor to its mature form in vitro. Next, we show that disruption of a gene encoding plastidic SPase I (Plsp1) resulted in the accumulation of immature forms of Toc75, severe reduction of plastid internal membrane development, and a seedling lethal phenotype. These phenotypes were rescued by the overexpression of Plsp1 complementary DNA. Plsp1 appeared to be targeted both to the envelope and to the thylakoidal membranes; thus, it may have multiple functions.

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Plsp1 is targeted to chloroplast membranes in vitro. (A) Radiolabeled precursors (tl; 10% input) were incubated with pea chloroplasts. Chloroplasts were either directly analyzed (imp) or lysed and separated into supernatant (s) and pellet (p) fractions as described previously (Inoue and Keegstra, 2003). Alternatively, chloroplasts containing imported proteins were treated without or with thermolysin or trypsin (0.1 μg protease/1 μg chlorophyll-equivalent chloroplasts) and separated into soluble (s) and pellet (p) fractions or treated with proteases with the presence of 1% Triton X-100 (imp/Tx). The translation products directly treated with proteases are also shown. Imported DGD1 and m110N are indicated with arrowheads in the bottom two panels. (B) Imported radiolabeled proteins (C) were further fractionated by centrifugation to thylakoids (T) or total membrane (T/E) and envelope (E) fractions as described previously (Baldwin et al., 2005). Black lines indicate grouping of images of different exposures from different gels (A) or that of images from different portions of the same gel (B).
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fig4: Plsp1 is targeted to chloroplast membranes in vitro. (A) Radiolabeled precursors (tl; 10% input) were incubated with pea chloroplasts. Chloroplasts were either directly analyzed (imp) or lysed and separated into supernatant (s) and pellet (p) fractions as described previously (Inoue and Keegstra, 2003). Alternatively, chloroplasts containing imported proteins were treated without or with thermolysin or trypsin (0.1 μg protease/1 μg chlorophyll-equivalent chloroplasts) and separated into soluble (s) and pellet (p) fractions or treated with proteases with the presence of 1% Triton X-100 (imp/Tx). The translation products directly treated with proteases are also shown. Imported DGD1 and m110N are indicated with arrowheads in the bottom two panels. (B) Imported radiolabeled proteins (C) were further fractionated by centrifugation to thylakoids (T) or total membrane (T/E) and envelope (E) fractions as described previously (Baldwin et al., 2005). Black lines indicate grouping of images of different exposures from different gels (A) or that of images from different portions of the same gel (B).

Mentions: Maturation of Toc75 appears to occur in the envelope membranes. Thus, if Plsp1 is directly responsible for this process, it should be located at the envelope membranes. Recently, however, Plsp1 was found in the thylakoidal membrane proteome (Peltier et al., 2004). To confirm its suborganellar localization, we subjected radiolabeled Plsp1 to in vitro chloroplast import assays. As shown in Fig. 4 A, the 32.6-kD Plsp1 precursor protein (lane 1) was processed to 25 kD (lane 2), which was integrated into membranes and was not exposed to the surface of the organelle, as indicated by its resistance to carbonate extraction (lane 6) and also to thermolysin treatment (lane 12). Trypsin is a protease that can reach the surface of the inner membrane (Jackson et al., 1998). Interestingly, trypsin treatment of chloroplasts containing the imported Plsp1 resulted in the production of a partially degraded protein of ∼22 kD along with the intact mature protein (Fig. 4 A, lane 18). The ratio of the two proteins was ∼1:1, which was consistent among three independent experiments and did not change by increasing the concentration of trypsin up to 10 times (not depicted). The Plsp1 precursor was completely degraded after direct incubation with trypsin (Fig. 4 A, lane 13). However, when chloroplasts containing the imported protein were treated with trypsin in the presence of a detergent, a 22-kD protein was produced (Fig. 4 A, lane 14). These data imply that after Plsp1 was incorporated into chloroplasts, it formed a structure in which most lysine and arginine residues in the protein were highly protected even when the lipid bilayer was disrupted. Under the current conditions, a peripheral inner membrane protein that was located in the intermembrane space, Tic22 (Kouranov et al., 1998), was recovered in the supernatant after alkaline treatment (Fig. 4 A, lane 23) and was completely digested by trypsin (Fig. 4 A, lane 36). An outer membrane protein, DGD1 (Froehlich et al., 2001), was susceptible to thermolysin, whereas a stroma-facing inner membrane protein, m110N (Jackson et al., 1998), was resistant to trypsin (Fig. 4 A, lanes 44 and 52, respectively). Together, these results suggest that Plsp1 was targeted to at least two subcompartments: at the location where trypsin can reach (i.e., at the envelope membranes) and at the location where trypsin cannot reach (i.e., at the thylakoid). This idea was supported by fractionation analysis (Fig. 4 B). Imported Plsp1 was detected mainly in the thylakoid but also in envelope fractions in a ratio of ∼6:1 (Fig. 4 B, lanes 1–4). In contrast, envelope proteins Toc75-IV and Tic110 were detected almost equally in the thylakoid and in envelope fractions (Fig. 4 B, lanes 5–12), and light harvesting chlorophyll a/b–binding protein was recovered in the thylakoid but not in the envelope fraction (Fig. 4 B, lanes 13–16).


Complete maturation of the plastid protein translocation channel requires a type I signal peptidase.

Inoue K, Baldwin AJ, Shipman RL, Matsui K, Theg SM, Ohme-Takagi M - J. Cell Biol. (2005)

Plsp1 is targeted to chloroplast membranes in vitro. (A) Radiolabeled precursors (tl; 10% input) were incubated with pea chloroplasts. Chloroplasts were either directly analyzed (imp) or lysed and separated into supernatant (s) and pellet (p) fractions as described previously (Inoue and Keegstra, 2003). Alternatively, chloroplasts containing imported proteins were treated without or with thermolysin or trypsin (0.1 μg protease/1 μg chlorophyll-equivalent chloroplasts) and separated into soluble (s) and pellet (p) fractions or treated with proteases with the presence of 1% Triton X-100 (imp/Tx). The translation products directly treated with proteases are also shown. Imported DGD1 and m110N are indicated with arrowheads in the bottom two panels. (B) Imported radiolabeled proteins (C) were further fractionated by centrifugation to thylakoids (T) or total membrane (T/E) and envelope (E) fractions as described previously (Baldwin et al., 2005). Black lines indicate grouping of images of different exposures from different gels (A) or that of images from different portions of the same gel (B).
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Related In: Results  -  Collection

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fig4: Plsp1 is targeted to chloroplast membranes in vitro. (A) Radiolabeled precursors (tl; 10% input) were incubated with pea chloroplasts. Chloroplasts were either directly analyzed (imp) or lysed and separated into supernatant (s) and pellet (p) fractions as described previously (Inoue and Keegstra, 2003). Alternatively, chloroplasts containing imported proteins were treated without or with thermolysin or trypsin (0.1 μg protease/1 μg chlorophyll-equivalent chloroplasts) and separated into soluble (s) and pellet (p) fractions or treated with proteases with the presence of 1% Triton X-100 (imp/Tx). The translation products directly treated with proteases are also shown. Imported DGD1 and m110N are indicated with arrowheads in the bottom two panels. (B) Imported radiolabeled proteins (C) were further fractionated by centrifugation to thylakoids (T) or total membrane (T/E) and envelope (E) fractions as described previously (Baldwin et al., 2005). Black lines indicate grouping of images of different exposures from different gels (A) or that of images from different portions of the same gel (B).
Mentions: Maturation of Toc75 appears to occur in the envelope membranes. Thus, if Plsp1 is directly responsible for this process, it should be located at the envelope membranes. Recently, however, Plsp1 was found in the thylakoidal membrane proteome (Peltier et al., 2004). To confirm its suborganellar localization, we subjected radiolabeled Plsp1 to in vitro chloroplast import assays. As shown in Fig. 4 A, the 32.6-kD Plsp1 precursor protein (lane 1) was processed to 25 kD (lane 2), which was integrated into membranes and was not exposed to the surface of the organelle, as indicated by its resistance to carbonate extraction (lane 6) and also to thermolysin treatment (lane 12). Trypsin is a protease that can reach the surface of the inner membrane (Jackson et al., 1998). Interestingly, trypsin treatment of chloroplasts containing the imported Plsp1 resulted in the production of a partially degraded protein of ∼22 kD along with the intact mature protein (Fig. 4 A, lane 18). The ratio of the two proteins was ∼1:1, which was consistent among three independent experiments and did not change by increasing the concentration of trypsin up to 10 times (not depicted). The Plsp1 precursor was completely degraded after direct incubation with trypsin (Fig. 4 A, lane 13). However, when chloroplasts containing the imported protein were treated with trypsin in the presence of a detergent, a 22-kD protein was produced (Fig. 4 A, lane 14). These data imply that after Plsp1 was incorporated into chloroplasts, it formed a structure in which most lysine and arginine residues in the protein were highly protected even when the lipid bilayer was disrupted. Under the current conditions, a peripheral inner membrane protein that was located in the intermembrane space, Tic22 (Kouranov et al., 1998), was recovered in the supernatant after alkaline treatment (Fig. 4 A, lane 23) and was completely digested by trypsin (Fig. 4 A, lane 36). An outer membrane protein, DGD1 (Froehlich et al., 2001), was susceptible to thermolysin, whereas a stroma-facing inner membrane protein, m110N (Jackson et al., 1998), was resistant to trypsin (Fig. 4 A, lanes 44 and 52, respectively). Together, these results suggest that Plsp1 was targeted to at least two subcompartments: at the location where trypsin can reach (i.e., at the envelope membranes) and at the location where trypsin cannot reach (i.e., at the thylakoid). This idea was supported by fractionation analysis (Fig. 4 B). Imported Plsp1 was detected mainly in the thylakoid but also in envelope fractions in a ratio of ∼6:1 (Fig. 4 B, lanes 1–4). In contrast, envelope proteins Toc75-IV and Tic110 were detected almost equally in the thylakoid and in envelope fractions (Fig. 4 B, lanes 5–12), and light harvesting chlorophyll a/b–binding protein was recovered in the thylakoid but not in the envelope fraction (Fig. 4 B, lanes 13–16).

Bottom Line: Next, we show that disruption of a gene encoding plastidic SPase I (Plsp1) resulted in the accumulation of immature forms of Toc75, severe reduction of plastid internal membrane development, and a seedling lethal phenotype.These phenotypes were rescued by the overexpression of Plsp1 complementary DNA.Plsp1 appeared to be targeted both to the envelope and to the thylakoidal membranes; thus, it may have multiple functions.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Sciences, College of Agricultural and Environmental Sciences, University of California, Davis, CA 95616, USA. kinoue@ucdavis.edu

ABSTRACT
The protein translocation channel at the plastid outer envelope membrane, Toc75, is essential for the viability of plants from the embryonic stage. It is encoded in the nucleus and is synthesized with a bipartite transit peptide that is cleaved during maturation. Despite its important function, the molecular mechanism and the biological significance of the full maturation of Toc75 remain unclear. In this study, we show that a type I signal peptidase (SPase I) is responsible for this process. First, we demonstrate that a bacterial SPase I converted Toc75 precursor to its mature form in vitro. Next, we show that disruption of a gene encoding plastidic SPase I (Plsp1) resulted in the accumulation of immature forms of Toc75, severe reduction of plastid internal membrane development, and a seedling lethal phenotype. These phenotypes were rescued by the overexpression of Plsp1 complementary DNA. Plsp1 appeared to be targeted both to the envelope and to the thylakoidal membranes; thus, it may have multiple functions.

Show MeSH
Related in: MedlinePlus